CN104090104B - Carbon nano-tube micro-cantilever biosensor for tumor-marker analyte detection that concentration is 0.5-10 mcg/ml - Google Patents

Abstract

A carbon nanotube microcantilever biosensor for the detection of tumor markers at a concentration of 0.5‑10µg/mL, including a support (1), a base material (2), a carbon nanotube (3), and a pick-up circuit (4); The material (2) is fixed on one side of the support (1) to form a micro-cantilever beam structure, the carbon nanotubes (3) are grown on the base material (2), and the pick-up circuit (4) is under the base material (2); The nucleic acid aptamer (5) attached to the carbon nanotube (3). The invention uses nucleic acid aptamers modified on carbon nanotubes to detect tumor markers. Using the microcantilever as the sensor platform for tumor marker detection can easily meet the requirements of high-throughput, miniaturization, and arraying for detection, and achieve the purpose of joint detection of multiple indicators of tumor markers. The micro-cantilever beam is made by MEMS processing technology and can be mass-produced, thereby reducing the cost of the device.

Description

Carbon nanotube microcantilever biosensor for tumor marker detection at a concentration of 0.5-10 μg/mL

technical field

The invention relates to the field of biomedical engineering, in particular to a carbon nanotube micro-cantilever biosensor.

Background technique

The method of using sensors to detect tumor markers has been reported, including mass and heat immunosensors, electrochemical immunosensors, etc. These sensors are generally only for the detection of a single tumor marker, the false positive rate is high, the detection accuracy is not enough, and it is easy to delay or aggravate the disease. It has become an urgent technical problem to develop new detection technology, improve positive rate and detection accuracy, and realize online detection.

The micro-cantilever sensor fixes the object under test and the micro-cantilever together in some way, and realizes signal conversion through the deflection displacement of the micro-cantilever or the change of the resonance frequency. The name is the method of micro-cantilever sensor using functionalized carbon nanotubes as sensitive materials, and the invention patent application with the publication number CN201010241824.X combines carbon nanotubes with micro-cantilever to realize the detection of specific gases. This method has high sensitivity and good repeatability; the disadvantage is that the carbon nanotubes need to be modified with functional groups according to the chemical properties of the target gas, and the operation is relatively complicated. When the concentration of tumor markers is 0.5-10 μg/mL, the microcantilever beam is used as the sensor platform for tumor marker detection, and the specific reaction between the nucleic acid aptamer and the tumor marker is used to form a complex. Nanotube micro-cantilever beam biosensors produce mass effect, and the technology of using this mass effect to realize the detection of tumor markers has not been reported yet.

Contents of the invention

The technical problem to be solved by the present invention is to provide a carbon nanotube microcantilever biosensor for the detection of tumor markers with a concentration of 0.5-10 μg/mL. The biosensor is small in size, simple in structure, easy to operate, and can realize online Multi-indicator detection.

In order to solve this technical problem, the present invention realizes the detection of tumor markers by modifying nucleic acid aptamers on carbon nanotubes.

The biosensor of the present invention includes a support, a base material, a pick-up circuit, carbon nanotubes, and nucleic acid aptamers on the carbon nanotubes. The base material is fixed on one side of the support to form a micro-cantilever structure; the pick-up circuit part is under the base material; the pick-up circuit part connects four piezoresistors into a Wheatstone bridge form; the carbon nanotube The carbon nanotube microcantilever sensor is formed on the top of the microcantilever; the nucleic acid aptamer is modified on the carbon nanotube by hydrophobic interaction, or modified on the carbon nanotube by π-π superposition; the nucleic acid aptamer The aptamer and the tumor marker form a complex through a specific recognition reaction, and the mass effect produced by the complex on the micro-cantilever biosensor is used to realize detection.

The invention uses the micro-cantilever beam as a sensor platform for detecting tumor markers, and manufactures a carbon nanotube micro-cantilever biosensor capable of detecting various tumor markers.

Description of drawings

Fig. 1 is a schematic diagram of a carbon nanotube microcantilever biosensor for tumor marker detection.

Advantages and features of the present invention

1. The present invention uses carbon nanotube micro-cantilever biosensors to detect tumor markers. The carbon nanotubes grown by LPCVD method have stable quality, are not easy to shift and deform, and are beneficial to the modification of subsequent processes to form stable detection probes. Modified by hydrophobic interaction or π-π superposition interaction, the nucleic acid aptamer is not easy to be lost on the carbon nanotubes, and works together with the LPCVD method to grow carbon nanotubes, which facilitates the formation of tumor markers and nucleic acid aptamers in subsequent steps The mass effect of the compound on the micro-cantilever is stable, thereby causing the stability of the resonance frequency change of the micro-cantilever, and laying the foundation for realizing the detection concentration range of tumor markers in the range of 0.5-10 μg/mL. The above-mentioned technical features support each other and work together to realize rapid and accurate detection when the concentration of tumor markers is 0.5-10 μg/mL, and the operation is simple.

2. Since carbon nanotubes are grown on the micro-cantilever beams, and nucleic acid aptamers are modified on the carbon nanotubes, the biosensor is small in size and simple in structure; it can realize online detection.

detailed description

The present invention will be described in further detail below in conjunction with the accompanying drawings and embodiments, but is not limited to the embodiments.

Example:

Referring to Figure 1, Figure 1 is a schematic diagram of a carbon nanotube microcantilever biosensor for tumor marker detection, including a bracket 1, a base material 2, a carbon nanotube 3, a pick-up circuit 4, and a nucleic acid adapter attached to the carbon nanotube 3 Body 5. The base material 2 is fixed on one side of the support 1 to form a micro-cantilever beam structure; the carbon nanotube 3 is grown on the base material 2; and the pick-up circuit 4 is under the base material 2; the carbon nanotube 3 is modified by a hydrophobic Layer aptamer 5.

The present invention is prepared and operated according to the following conventional processes

1. Fabrication of micro-cantilever structure

The micro-cantilever is processed into a micro-cantilever structure by using semiconductor material silicon as the base material 2 .

2. Fabrication of pickup circuit 4

For the pick-up circuit, silicon varistors are fabricated on the lower surface of the base material 2 by microelectronic technology, and four varistors are connected to form a Wheatstone bridge.

3. Cantilever beam growth and carbon nanotube coating process

The upper surface of the base material 2 in the preceding steps is cleaned by ultrasonic cleaning with acetone, absolute ethanol, and deionized water respectively, and then carbon nanotubes are grown by low-pressure chemical vapor deposition (LPCVD). Carbon nanotubes can also be coated on silicon substrates by pyrolysis, coating, or other methods.

4. Modification of nucleic acid aptamers on carbon nanotube microcantilevers

The nucleic acid aptamer is modified on the carbon nanotube through hydrophobic interaction to form a detection probe that can specifically recognize tumor markers, thereby constructing the carbon nanotube micro-cantilever biosensor.

The steps of the present invention for tumor marker detection are as follows:

(1) Fabricate detection probes containing tumor marker nucleic acid aptamers on carbon nanotube microcantilevers;

(2) Put the detection probe into the sample to be tested, the concentration of the tumor marker in the sample is 0.5-10 μg/mL, and the tumor marker in the sample to be tested is complexed with the nucleic acid aptamer on the detection probe through a specific reaction and attached to the microcantilever.

(3) The mass of the formed complex is positively correlated with the concentration of the tumor marker in the sample to be tested.

(4) The mass change of the complex on the micro-cantilever causes the flexural displacement or the change of the resonance frequency of the micro-cantilever, thereby realizing the detection of tumor markers.

Claims (1)

1. the detection method of a tumor markers non-diagnostic purpose, it uses carbon nano-tube micro-cantilever biosensor to detect, described carbon nano-tube micro-cantilever biosensor includes support (1), silicon substrate material (2), CNT (3), pick-up circuit (4)；Described silicon substrate material (2) is fixed on support (1) side and constitutes micro cantilever structure；Described CNT (3) is grown in above silicon substrate material (2), the first upper surface at silicon substrate material (2) is used to be carried out processing, ultrasonic waves for cleaning is carried out, then with Low Pressure Chemical Vapor Deposition (LPCVD) growth CNT (3) respectively with acetone, dehydrated alcohol, deionized water；Described pick-up circuit (4), below silicon substrate material (2), is the piezoresistive effect utilizing silicon, and four varistors are connected into wheatstone bridge form；It is additionally included in CNT (3) and is modified with one layer of aptamer (5) above with hydrophobic interaction or π-π superposition；It is pyrolysismethod that described CNT (3) is grown in the growing method of silicon substrate material (2), or cladding process；

Detecting step is as follows:

A, first make containing the detection probe of tumor markers aptamer in carbon nano-tube micro-cantilever；

B, putting in sample to be tested by detection probe, in sample to be tested, tumor markers is formed complex by specific reaction with the aptamer on detection probe and is attached on micro-cantilever；

C, the quality size of the complex formed are proportionate with tumor-marker substrate concentration in sample to be tested；

The mass change that D, described complex produce on micro-cantilever causes the change of micro-cantilever flexing displacement or resonant frequency, thus realizes the detection to tumor markers.